Increased intracellular content of enteroglucagon in proximal colon is related to intestinal adaptation to germ-free status

Changes in the frequency of endocrine cells are evidence of intestinal adaptation to germ-free (GF) status. Not only the distribution of these cells along the intestine, but also the differences in intracellular content of these regulatory peptides may be explored to explain functional and structural aspects of GF intestinal adaptation. Focusing on the endocrine L-cells, we analyzed the intracellular content of enteroglucagon (EG) and peptide YY (PYY) throughout the intestine of the 14 GF and 14 conventional (CV) mice by using immunohistochemistry and the supra-optimal dilution technique. The percentage of EG-immunoreactive cells, but not of PYY-immunoreactive cells stained at supra-optimal dilution was significantly higher in the proximal colon of GF mice than in the CV counterparts (P < 0.05). Since the content of co-stored PYY did not differ between GF and CV mice, the higher content of EG was compatible with a selective cellular response. Moreover, in the cecum of GF mice, the density of EG-immunoreactive cells was significantly higher than that of PYY-immunoreactive cells (P < 0.05). These results are consistent with preferential production of EG by L-cells at the expense of PYY in the proximal colon and in the enlarged cecum of GF mice. In addition, they may reflect the dynamics of the GF intestinal epithelium and/or be correlated with the higher serum levels of these peptides. The role of endocrine cells needs to be better studied in human and other experimental adaptative conditions in order to elucidate the regulatory mechanisms of intestinal functions.     

Saccharomyces cerevisiae strain UFMG 905 protects against bacterial translocation, preserves gut barrier integrity and stimulates the immune system in a murine intestinal obstruction model

Probiotic is a preparation containing microorganisms that confers beneficial effect to the host. This work assessed whether oral treatment with viable or heat-killed yeast Saccharomyces cerevisiae strain UFMG 905 prevents bacterial translocation (BT), intestinal barrier integrity, and stimulates the immunity, in a murine intestinal obstruction (IO) model. Four groups of mice were used: mice undergoing only laparotomy (CTL), undergoing intestinal obstruction (IO) and undergoing intestinal obstruction after previous treatment with viable or heat-killed yeast. BT, determined as uptake of ^99mTc-E. coli in blood, mesenteric lymph nodes, liver, spleen and lungs, was significantly higher in IO group than in CTL group. Treatments with both yeasts reduced BT in blood and all organs investigated. The treatment with both yeasts also reduced intestinal permeability as determined by blood uptake of ^99mTc-DTPA. Immunological data demonstrated that both treatments were able to significantly increase IL-10 levels, but only viable yeast had the same effect on sIgA levels. Intestinal lesions were more severe in IO group when compared to CTL and yeasts groups. Concluding, both viable and heat-killed cells of yeast prevent BT, probably by immunomodulation and by maintaining gut barrier integrity. Only the stimulation of IgA production seems to depend on the yeast viability.     

Co-Expression of p16, Ki67 and COX-2 Is Associated with Basal Phenotype in High-Grade Ductal Carcinoma In Situ of the Breast

We assessed the co-expression of cell cycle-related biomarkers in a series of 121 consecutive cases of high-grade ductal carcinoma in situ (DCIS), pure or associated with invasive carcinoma, and their associations with the different immunoprofiles of DCIS. Cases were identified from the histopathology files of the Breast Pathology Laboratory, Federal University of Minas Gerais, Brazil, from 2003 to 2008. The expression of estrogen receptor, progesterone receptor, HER2 overexpression, cytokeratin 5, epidermal growth factor receptor 1, cyclooxygenase-2, p16 and Ki67 were assessed. Tumors were placed into five subgroups according to their immunohistochemical profile: luminal A, luminal B, HER2, basal-like and “not classified”. We found that the basal phenotype was associated with a higher frequency of p16-positive cases (83%) and the luminal A phenotype showed a higher frequency of p16-negative cases (93%; p=0.000). The association of biomarkers p16⁺/Ki67⁺/COX2⁺ was expressed in 02/06 cases (33.3%) of the basal phenotype but in only 01/70 cases (1.4%) of the luminal A phenotype (p=0.01). The co-expression of p16⁺/Ki67⁺/COX2^- was associated with a basal phenotype (p=0.004). P16 expression, p16⁺/Ki67⁺/COX2⁺ and p16⁺/Ki67⁺/COX2^- co-expression showed significant associations with the basal phenotype and these profiles could be used to guide more aggressive treatment strategies in patients with high-grade DCIS.     

Modeling population dynamics and conservation of arapaima in the Amazon

To promote understanding of fish population dynamics in tropical river-floodplains, we have synthesized existing information by developing a largely empirical population model for arapaima (Arapaima sp.). Arapaima are characterized by very large bodies, relatively late sexual maturity, small clutches, and large parental investment per offspring, and their populations are overexploited and even declining due to overfishing. We used unparalleled time series data on growth, reproduction, catch-at-age, and size-class abundance estimates for a population that has increased several-fold and undergone drastic changes in fishing practices in the Amazon, Brazil. Model population numbers were close to observed numbers, with generally low mean absolute percentage errors for juveniles (16%), adults (30%), and catch (18%). In using the model to test ecological hypotheses and to investigate management strategies, we found the following: (1) Annual recruitment is directly and positively related to spawner abundance, and it appears to be density-compensatory following a Beverton–Holt relation (R ² = 0.85). (2) Fishing-selectivity of arapaima caused by use of harpoons and gillnets can lower yield potentials dramatically through removal of the faster-growing individuals of the population. That is in part because fewer individuals live long enough to reproduce and survivors take longer to reach reproductive age. (3) Arapaima populations can sustain annual catches of up to 25% of the number of adults in the population the previous year if minimum size (1.5 m) and closed season (December–May) limits are met. (4) When 25% of the number of adults in the population the previous year is harvested under a 1.6 m minimum size limit of catch, catches are slightly smaller but abundance of adults in the population is considerably greater than under a 1.5 m limit. These findings can be used in ongoing management initiatives, but caution is needed because of present biological and ecological uncertainty about these fishes.     

<Emphasis Type="Italic">Rubus rosaefolius</Emphasis> Extract as a Natural Preservative Candidate in Topical Formulations

Even though the synthetic preservatives may offer a high antimicrobial efficacy, they are commonly related to adverse reactions and regarded as having potentially harmful effects caused by chronic consumption. The development of natural preservatives provides a way of reducing the amount of synthetic preservatives normally used in pharmaceutical and cosmetic preparations. In addition, these agents have less toxic effects and represent a possible natural and safer alternative of the preservatives. The purpose of this research was to evaluate the Rubus rosaefolius Smith extract efficiency as a natural preservative in base formulations. Of the extract, 0.2% (w/w) was assayed for its effectiveness of antimicrobial protection in two different base formulations (emulsion and gel). The microbial challenge test was performed following the standard procedures proposed by The United States Pharmacopoeia 33nd, European Pharmacopoeia 6th, Japanese Pharmacopoeia 15th, and the Cosmetics, Toiletries, and Fragrance Association using standardized microorganisms. The results demonstrated that R. rosaefolius extract at the studied concentration reduced the bacterial inocula, satisfying the criterion in all formulations, even though it was not able to present an effective preservative behavior against fungi. Thus, the investigation of new natural substances with preservative properties that could be applied in pharmaceutical and cosmetic products is relevant due to the possibility of substituting or decreasing the concentration of synthetic preservatives, providing a way for the development of safer formulas for the use of consumers.     

A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7_XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7_XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7_XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7_XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.     

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.     

Fatty acid composition of the tropical lichen Teloschistes flavicans and its cultivated symbionts

Fatty acid components, in both the free and combined form of the intact tropical lichen Teloschistes flavicans, and its isolated photobiont and mycobiont, were analyzed by GC-MS of derived methyl esters. Its rDNA analysis confirmed that the isolated cultured symbionts belong to the genera Trebouxia and Teloschistes, respectively. The fatty acid composition of the lichen did not correspond to those found in the isolated symbionts, suggesting that the fatty acid metabolism is markedly influenced by the symbiosis. Differences in the fatty acid composition in the lichen were observed during the summer (27 degrees C), when the main fatty acids were saturated and in the winter (22 degrees C) when an increase of unsaturated fatty acids occurred. Similar differences of composition were also observed for the cultured mycobiont at different temperatures. The increase in the unsaturation level at low temperatures would maintain the membrane fluidity. Our results are the first on the fatty acids of a tropical lichen and suggest that it is sensitive to small temperature variations, which influences its saturated and unsaturated fatty acid composition.     

Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms

The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M. frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation, M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85+/-0.6% inhibition after a 15 min incubation at 5 microg of venom/mL) and the response to acetylcholine (ACh; 55 or 110 microM), without affecting the response to KCl (13.4 mM). In mouse phrenic nerve-diaphragm preparation, the venom (5 microg/mL) produced a complete inhibition of the indirectly elicited contractile response after 50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-L-glutamate uptake in brain synaptosomes in a Ca2+-, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(beta-aminoethyl ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-L-glutamate uptake. M. lemniscatus carvalhoi venom cross-reacted with postsynaptic alpha-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies. It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of molecular targets on cell membranes.